Fused genes and their use for determining the presence of metals or of xenobiotic compounds

ABSTRACT

The invention relates to a fused gene containing: the promoter sequence of (a) gene(s) encoding the resistance to one or several metal(s) or encoding the catabolism of one or several xenobiotic compound(s), said promoter being inducible in the presence of said metal(s) or xenobiotic compound(s), or both, and downstream the promoter, a gene producing a detectable signal such as light emitting gene, said gene being under the control of said promoter, said gene producing a detectable signal being located at a position such that the induction of the promoter causes the transcription of the gene producing a detectable signal and such that there is no terminator between the promoter and the gene producing a detectable signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a file wrapper continuing application of U.S.Ser. No. 08/108,754 filed Dec. 27, 1993 now abandoned, application Ser.No. 08/108,754 is a national stage application of PCT application Ser.No. PCT/EP92/00445 filed 28 Feb. 1992 and published as WO 92/15687 on 17Sep. 1992.

The invention relates to fused genes, vectors containing them, processfor preparing them and their use for determining the presence of metalsor of xenobiotic compounds.

Toxical wastes are a significant contamination problem for a range ofindustries.

Among the substances involved, one may cite heavy metals and xenobioticcompounds which are very polluting and which may endanger health. Thesources of pollution are varied. Moreover, with the enforcement ofstrict regulations, in order to limit the wastes containing metals suchas heavy metals and xenobiotic compounds, there is a need for methods ofdetection of metals and xenobiotic compounds in environment.

Most of the methods used routinely to measure metal concentrations arephysical methods which rely on the substantial physical (usuallyelectronic) differences between the metal and the carrier medium.

Among these methods, the most commonly used are the inductively coupledplasma systems, the X-ray fluorescence or the atomic absorption.

The main advantage of these methods is the very low limits of detection(about 0,1 ppm) as well as a multielementary aspect of the analysis.But, the main drawbacks are the high price of the equipment, the use ofwhich requires high qualified people, the long time required forpreparing the samples to be analyzed and the sensitivity of thesemethods related to many interferences due to the nature of the samples.

Many organisms can tolerate high concentrations of heavy metals such ascadmium and lead. The mechanism involved varies. Specific, geneticallycoded resistance to heavy metals can evolve in populations of organismsexposed over long periods of time to heavy metals ("Genetic adaptationto heavy metals in aquatic organisms: a review" P. L. Klerks, J. S. Weis(1987), Environmental pollution 45: 173-205). Searches of soil in sitesheavily contaminated with heavy metals routinely reveal strains ofmicroorganisms with enhanced abilities to tolerate heavy metals. Severalsuch strains have been isolated from a heavily contaminated site inBelgium, and the genetics of their responses to heavy metals have beenanalyzed ("Alcaligenes eutrophus CH34 is a facultative chemilithotrophwith plasmid-bound resistance to heavy metals" M. Mergeay, D. Nies, H.G. Schlegel, J. Gerits, P. Charles, F. van Gijsegem (1985), J.Bacteriol. 162: 328-334; "Cloning of plasmid genes encoding resistanceto cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34" D. Nies, M.Mergeay, B. Friedrich, H. G. Schlegel (1987), J. Bacteriol.169:4865-4868).

Most microorganisms can degrade a wide variety of compounds to generatemetabolic energy and to make available metabolic intermediates, andparticularly carbon, for their use. Some organisms specialize in thedegradation of exotic materials, using unusual enzyme systems to do so.These are frequently soil bacteria that have evolved in sites whereindustrial activity has released a substantial amount of such materialinto the soil. The ability to degrade highly conjugated aromatichydrocarbons and their halide derivatives is a good example, as thesematerials are rarely found in nature and require special enzymes toinitiate their degradation, usually by oxygenation.

Alcaligenes eutrophus, as a bacterial organism, presents specificinducible genes of resistance with respect to heavy metals or involvedin the catabolism of xenobiotics such as PCBs.

The bacteria of the group of Alcaligenes eutrophus (gram negative) havebeside the property of being facultative chemilithotroph, the propertyof comprising one of several megaplasmids which confer on them multipleresistances with respect to heavy metals. These bacteria have beendiscovered in the neighborhood of non ferrous metal facturies and in theneighborhood of mining sites in Belgium and in Zaire (Diels et al.,1988(a), Isolation and characterization of resistant bacteria to heavymetals from mining areas of Zaire. Arch. Int. Physiol. Biochim. 96(2)B83; Diels et al., 1988(b), Detection of heterotrophic bacteria withplasmid-bound resistances to heavy metals from Belgian industrial sites.Arch. Int. Physiol. Biochim. 96(2)B84).

Alcaligenes eutrophus CH34 (ATCC 43123) presents two megaplasmids:pMOL28 (165 kb) and pMOL30 (240 kb). pMOL30 has been found to beinvolved in the expression of heavy metal resistance to cadmium, zinc,cobalt, copper, lead, mercury, thallium and manganese. pMOL28 has beenfound to be involved in the expression of heavy metal resistance tocobalt, chromium, thallium and mercury (Mergeay et al., 1985,Alcaligenes eutrophus CH34 is a facultative chemilithotroph withplasmid-bound resistance to heavy metals. J. Bacteriol. 169, 328-334;Nies et al., 1987, Cloning plasmid genes coding resistance to cadmium,zinc and cobalt in Alcaligenes eutrophus CH34. J. Bacteriol. 169,4865-4868; Diels et al. 1989(a), Large plasmid governing multipleresistance to heavy metals: a genetic approach. Toxicol. Environ. Chem.23, 79-89).

These megaplasmids are transmissible by homologous crossings (Mergeay etal., 1985, Alcaligenes eutrophus CH34 is a facultative chemilithotrophwith plasmid-bound resistance to heavy metals. J. Bacteriol. 169,328-334, Table 1).

The restriction map of the native plasmids of Alcaligenes eutrophus, thelocus of the various resistance with respect to heavy metals as well asresistance mechanism for some metals (mercury, cadmium, zinc, nickel,cobalt, chromium) start to be understood.

pMOL30, for instance, contains an EcoRI fragment of 9,1 kb which isnamed czc, which has been evidenced by cloning and which conferssimultaneously a resistance to cadmium, zinc and cobalt ions (Nies etal., 1987, Cloning plasmid genes coding resistance to cadmium, zinc andcobalt in Alcaligenes eutrophus CH34. J. Bacteriol. 169, 4865-4868).

In the case of cadmium, cobalt, nickel and zinc, the resistance isdetermined by an efflux system (expulsion of the metallic cations aftertheir entry into the cell). Besides, an accumulation of the metal seemsto take place at the level of the bacterial envelops further to analcalinisation of the culture medium by the bacteria themselves (Dielset al., 1989(b), Accumulation of Cd and zinc ions by Alcaligeneseutrophus strains. Biohydrometallury 89. Jackson Hale USA). Thisphenomenon of accumulation takes place at the stationary phase anddepends on the conditions of metabolism.

Gene and protein fusions have been instrumental in the study of generegulation, protein processing, export and other aspects of genefunction.

All reporter gene systems in current use involve genes that encode anenzymatically active protein. The sensitivity of these systems variesaccording to the properties of the reporter enzyme, the nature andquality of the available assays and the presence or absence ofinterfering activities in the cell type. The lactose (lac) operon ofEscherichia coli has been employed most extensively in these studiesbecause a great amount of information is available regarding variousaspects of this genetic system (Berman M. L. 1983, "Vectors forconstructing hybrid genes" Biotechniques 1:178-183; Koenen et al. 1982,"Immunoenzymatic detection of expresses gene fragments coled in the lacZgene of E. coli. J. Bacteriol. 1:509-512; Silhavy et al., 1985, Uses oflac fusions for the study of biological problems. Microbiol. Rev. 49,398-418; Silhavy et al., 1984, "Experiments with gene fusions" ColdSpring Harbor Laboratory. Cold Spring Harbor, N.Y.).

A number of plasmid vectors have been designed for the purpose ofcloning and the subsequent evaluation of lac gene with promoter activity(Casadaban et al., 1980, "In vitro gene fusions that join anenzymatically active beta-galactosidase segment to amino-terminalfragments of exogenous proteins: Escherichia coli plasmid vectors forthe detection and cloning of translational initiation signals" J.Bacteriol. 143:971-980; Shapira et al., 1983, "New versatile plasmidvectors for expression of hybrid proteins coded by a cloned gene fusedto lacZ gene sequences encoding enzymatically active carboxyterminalportion of beta-galactosidase" Gene 25:71-82; Minton N. P., 1984,"Improved plasmid vectors for the isolation of translational lac genefusions" Gene 31:269-273) or for the study of protein function. Theyutilize gene transcription and translation initiation signals and resultin enzymatically active β-galactosidase proteins containingamino-terminal amino acid sequences from the exogenous gene (Muller-Hillet al., 1976, "Repressor-galactosidase chimaeras in Markam R. and HorneR. W. (eds) Structure-function relationship of proteins" North-Holland,New York, pp. 167-179; Bassford et al., 1978, "Genetic fusions of thelac operon: a new approach to the study of biological process in MillerJ. H. and Reznikoff W. S. (eds) The operon" Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 245-261; Guarente et al.,1980, "Improved methods for maximizing expression of a cloned gene:bacterium that synthesizes rabbit beta-globin" Cell 20:543-553).

These hybrid proteins have been purified readily by following theirβ-galactosidase activity and used for determining amino-terminalfunctional domains of proteins (Muiller-Hill et al., 1976,"Repressor-galactosidase chimaeras in Markam R. and Horne R. W. (eds)Structure-function relationship of proteins" North-Holland, New York,pp. 167-179; Silhavy et al., 1976, "Conversion of beta-galactosidase toa membrane-bound state by gene fusion" Proc. Natl. Acad. Sci. USA73:3423-3427; Hall M. et al., 1981, "Gene analysis of the major outermembrane proteins of Escherichia coli in Roman H. L., Campbell A., andSandler L. M. (eds)" Annual Reviews of Genetics, vol. 15, AnnualReviews, Palo Alto Calif. 91-142) and for eliciting antibody formationagainst amino-terminal antigenic determinants (Schuman et al., 1988,"Labeling of proteins with beta-galactosidase by gene fusionidentification of a cytoplasmic membrane component of Escherichia colimaltose transport system" J. Chem. 225:168-174).

Another reporter gene is the luciferase reporter gene. Bacterialluciferase enzymes catalyze a light emitting reaction in luminousbacteria. The light emitting luciferase catalyzed reaction is asfollows:

    RCHO+O.sub.2 +FMNH.sub.2 →RCOOH+FMN+H.sub.2 O+photon (490 nm)

in which R is an aliphatic moiety containing at least seven carbonatoms, preferably from 7 to 14 carbon atoms, FMN is a flavinmononucleotide and FMNH₂ is reduced flavin mononucleotide (Meighen E.A., 1988, "Enzymes and genes from the lux operon of bioluminescentbacteria" Ann. Rev. Microbiol. 42:151-176).

In bacteria, the oxidized flavin is efficiently reduced and continuouslyavailable to cytoplasmic enzymes, such as luciferase.

Upon external addition of the aldehyde substrate, which instantlypenetrates living cells, the activity of luciferase can be followed invivo by measuring light emission. Light can be monitored by a number ofmethods and with high sensitivity. Since a bacterial luciferase moleculegives rise to about one photon in the luciferase reaction, as little as10 luciferase molecules can be detected by a luminometer (Olsson O. etal., 1988, "The use of the luxA gene of the bacterial luciferase operonas a reporter gene", Mol. Gen. Genet. 215:1-9).

The luciferase gene cluster from the marine microorganisms Vibriofischeri, the luxAB structural genes from V. harveyi and the fireflycDNA from Photinus pyralis have recently been introduced as reportergenes in procaryotic (Engebrecht J. et al., 1985, "Measuring geneexpression with light" Sciences 227:1345-1347; Legocki R. P. et al.,1986, "Bioluminescence in soybean root nodules: Demonstration of ageneral approach to assay gene expression in vivo by using bacterialluciferases" Proc. Natl. Acad. Sci. USA 83:9080-9084; Karp M. T. et al.,1986, "Continous in vivo monitoring of gene expression using clonedbacterial luciferase genes" Biolum. Chemil. pp. 385-389; Schmetterer G.et al., 1986, "Expression of luciferases from Vibrio harveyi and Vibriofischeri in filamentous cyanobacteria. J. Bacteriol. 167:411-414; CarmiO. A. et al., 1987, "Use of bacterial luciferases to establish apromoter probe vehicle capable of non destructive real-time analysis ofgene expression in Bacillus" spp. J. Bacteriol. 169:2165-2170; NussbaumA. et al., 1989, "Use of a bioluminescence gene reporter for theinvestigation of a red-dependent and gram-dependent plasmidrecombination i: Escherichia coli K12" J. Mol. Biol. 203:402) as well asin eucaryotic organisms (Ow D. W. et al., 1986, "Transcient and stableexpression of the firefly luciferase gene in plant cells and transgenicplants" Science 234:856-859; Dewet J. R. et al., 1985, "Cloning offirefly luciferase cDNA and expression of active luciferase in E. coli"Proc. Natl. Acad. Sci. USA 82:7870-7873; Williams T. M. et al., 1989,"Advantages of firefly luciferase as reporter gene: application to theinterleukin-2 gene promoter", Anal. Biochem. 176:28-32; Riggs C. D. etal. 1987, "Luciferase reporter gene cassettes for plant gene expressionstudies" Nucleic Acids Res. 15:8115; Dilella G. A. et al., 1987,"Utility of firefly luciferase as reporter gene for promoter activity intransgenic mice" Nucl. Acids Res. 16:4159).

The firefly luciferase enzyme catalyses the ATP-dependent oxidation of ahigh molecular weight substrate, luciferin (Deluca et al., 1978,Purification and properties of firefly luciferase. Methods Enzymol. 57,3-15; Mc Elroy et al., 1985, Firefly luminescence, p. 387-399 in J. G.Burr (ed.) Chemibioluminescence, Marcel Dekker Inc., New York). Thissubstance is only slowly transported through cell membranes, in contrastto the aldehyde substrate in the bacterial reaction.

The Journal of Biotechnology (September 1990, p.4749-4757, Burlage,Sayler and Larimer) describes the fusion of the lux genes of Vibriofischeri to a fragment from plasmid NAH7, containing the promoter forthe upper pathway of degradation of naphthalene (related to somenaturally occuring compounds) and the first three cistrons of the nahAgene. A Pseudomonas strain (gram negative bacterium) containing thisconstruction is inducible to high levels of light production in thepresence of a suitable substrate.

Molecular Biology (1989), 3(8), p. 1011-1023, describes the coupling ofthe proU to luxAB, proU being the promoter of a gene regulatingosmolarity in Salmonella typhimurium; the above plasmid thus obtained iscloned in E. coli and is used to monitor in vivo real time kinetics ofproU induction following osmotic shock.

The aim of the invention is to provide with a process for detecting thepresence of metals or xenobiotic compounds said process being sensitive,cheap, simple and being suitable for an automatic or field use.

The aim of the invention is to provide with a method for detecting thepresence of metals or xenobiotic compounds, requiring no expensive andno massive capital equipment and low operator intervention.

The aim of the invention is also to provide with a method enabling togive a positive reply (light emission) in the presence of a metal or axenobiotic compound.

Another aim of the invention is to provide with a method for detectingthe presence of metals and/or xenobiotic compounds which is specific forthe metal or the xenobiotic compound which is to be detected.

The invention relates to a fused gene containing:

the promoter sequence of (a) gene(s) encoding the resistance to one orseveral metal(s) or encoding the catabolism of one or several xenobioticcompound(s), said promoter being inducible in the presence of saidmetal(s) or xenobiotic compound(s), or both,

and downstream the promoter, a gene producing a detectable signal suchas light emitting gene, said gene being under the control of saidpromoter, said gene producing a detectable signal being located at aposition such that the induction of the promoter causes thetranscription of the gene producing a detectable signal and such thatthere is no terminator between the promoter and the gene producing adetectable signal, said gene being such that it enables to recycle fattyacid (which has been generated during the reaction responsible for thedetectable signal) into aldehyde.

The expression "the gene producing a detectable signal being under thecontrol of the promoter" means that the promoter of the gene producing adetectable signal has been deleted.

By metal, one designates the transition metals, the rare earth, theelements having metallic properties in the families IIIa, IVa, Va andVIa of the Mendelieff table.

By metals, one may cite for example cadmium, zinc, cobalt, copper, lead,mercury, thallium, chromium and manganese under the form of salts,either in a soluble or non soluble state.

The expression "inducible promoter in the presence of said metal" meansthat there is a minimum concentration of said metal under which thepromoter is not induced. This depends particularly upon the nature ofthe promoter region and its regulation, the accessibility of the metalto the promoter region, the nature and the solubility of the metal.

The xenobiotic compounds designate the compounds which may endangerhealth and which are man made chemicals (non naturally occurringcompounds). By way of example, one may cite fungicides, herbicides,pesticides, insecticides, chloroorganic compounds, particularly biphenylcompounds.

In the following, the expression "resistance gene" corresponds to thegene responsible for the resistance to one or several metals and theexpression "catabolism gene" corresponds to the gene responsible for thecatabolism of one or several xenobiotic compounds.

The fused genes of the invention are placed in a host cell, e.g.bacteria, for the production of light to occur.

In the bioluminescent cell, the reaction of light production takes placewith the oxidation of long chained aldehydes and of reducedmononucleotide flavine (FMNH₂). The energy source of this reaction isgiven by the transformation of aldehyde (RCHO) into its correspondingfatty acid (RCOOH) according to the following reaction:

    RCHO+FMNH.sub.2 +O.sub.2 →RCOOH+FMN+H.sub.2 O+hυ.

RCHO representing an aldehyde from 7 to 14 carbon atoms.

The reaction always occurs because there is always a small amount ofaldehyde in the host cell.

When there is no more aldehyde in the host cell, it is necessary to addextra aldehyde, to obtain the production of light. However, aldehyde hasthe drawback of being toxic and besides, in this system, there isaccumulation of fatty acid, which is stored by the host cell and istoxic in the long run.

Besides, the light production depends on the added exogenous aldehyde.

In order to avoid these drawbacks, the gene which produces thedetectable signal is such that it is liable to recycle fatty acid intoaldehyde according to the following reaction:

    RCOOH+NADPH.sub.2 +ATP→RCHO+NADP+AMP+PPi.

This avoids the use of exogenous aldehyde and this prevents fatty acidfrom being accumulated in the host cell.

It may be possible to make a luminescence test which responds to severalanalytes with different signals. The Vibrio fischeri genes (giving greenlight) could be used to detect an analyte (for example: a metal) and adifferent luciferase gene producing light of slightly differentwavelengths (for instance: lux beetle luciferese which gives red light)in another fusion would detect another analyte (for example: axenobiotic or another metal). If more genes have different signals, itwould be possible to distinguish, in principle, the different analyteswithin the same bacteria.

According to another embodiment of the invention, the fused genecontains beside the inducible promoter also the coding sequence of thegene responsible for the resistance to one or several metals orresponsible for the catabolism of one or several xenobiotic compounds.

When the fused gene does not contain the coding sequence of the generesponsible for the resistance to one or several metals or responsiblefor the catabolism of one or several xenobiotic compounds, thisembodiment is very sensitive to metal or xenobiotics.

When the fused gene contains the coding sequence of the gene responsiblefor the resistance to one or several metals or responsible for thecatabolism of one or several xenobiotic compounds, the embodiment isless sensitive to metal or xenobiotics, but enables to measureconcentrations of metal or xenobiotics higher than the lethal ones.

In this case (i.e. when the coding part of the gene responsible for theresistance of a metal--or for the catabolism of a xenobiotic--ispresent), there might be translation of the resistance gene or of thecatabolism gene, if the translation machinery can be operated in thehost cell.

Translation of the resistance gene or of the catabolism gene might berequired if the concentration of the metal or of the xenobiotic compoundto be measured is higher than the lethal concentration of said metal orof said xenobiotic compound for the host cell containing the fused gene.

The invention also relates to a fused gene wherein the gene producing adetectable signal

either is located downstream the promoter and upstream the gene encodingthe resistance or the catabolism,

or is located downstream the promoter and downstream the gene encodingthe resistance or the catabolism,

or is located downstream the promoter and in the gene encoding theresistance or the catabolism.

When the fused gene contains only the inducible part of the resistancegene or of the catabolism gene without the coding sequence of said gene,the gene producing the detectable signal is downstream the promoter, andcan be spaced by a base pair sequence the length of which is such thatthe gene producing a detectable signal is still induced by the promoter.

When the fused gene contains, beside the promoter of the resistance geneor of the catabolism gene, also the coding part of said gene, the geneproducing the detectable signal can be

downstream the promoter and upstream the gene encoding resistance orcatabolism (i.e. between the inducible promoter and the coding part ofthe resistance gene or catabolism gene),

or can be downstream the coding part of the resistance gene or of thecatabolism gene,

or within the gene encoding resistance or catabolism.

When the gene producing the signal is located downstream the coding partof the gene encoding resistance or catabolism, the promoter must bestrong enough to provoke the transcription of the gene producing thesignal.

The strength of a promoter is defined by the ability to induce thetranscription of genes which are remote from the promoter.

When the gene producing the signal is located within the coding part ofthe resistance gene, or of the catabolism gene, there might betranscription and translation of the resistance gene, or of thecatabolism gene, if the resistance gene, or catabolism gene, is notdamaged by the insert containing the gene producing the signal, or,there might be partial transcription and partial translation of theresistance gene or of the catabolism gene.

When the gene producing the signal is located within the induciblepromoter, the gene producing the signal is no more under the control ofthe inducible promoter.

The invention also relates to a fused gene wherein a terminationsequence is located immediately upstream the promoter.

This termination sequence enables to avoid any interferencetranscription by other upstream promoters and would increase the ratiosignal/noise by lowering the expression of the light emitted by thebacteria in the absence of metals or of xenobiotic compounds.

According to an advantageous embodiment of the invention, the geneproducing a detectable signal is the luciferase gene.

Luciferase is interesting for the following reasons:

1) extremely low levels of light can be accurately measured, and lightcan be quantified linearly over many orders of magnitude;

2) there is no significant endogenous background activity (as there iswith β-galactosidase, for example)

3) transcription can be monitored non-invasively over time in vitro, inliquid or in a natural habitat, because the repeated application of thesubstrate (luciferin or n-decanal) is generally non-toxic;

4) the assays are very simple and inexpensive;

5) Light does not diffuse or accumulate in situ; the source of geneexpression can be localized spatially with high resolution.

The luciferase gene can originate from Vibrio fischeri or from Vibrioharveyi or from Photobacterium phosphoreum or from Xenorhabdusluminescens.

A preferred luciferase gene is the one originating from Xenorhabdusluminescence, cloned in E. coli (Frackman S. et al., 1990, "Cloning,Organization and expression of the bioluminescence genes of Xenorhabdusluminescens" J. Bact. 172:5767-5773).

A preferred luciferase gene is the one originating from V. fischeri; thesequence which is responsible for regulation as well as the expressionof bioluminescence as well as the synthesis of enzymes implied in thebioluminescence are known (see Devine et al., 1988, Nucleotide sequenceof the lux R and lux I genes and structure of the primary regulatoryregion of lux regulon of V. fischeri ATCC 7744. Biochem. 27, 837-842;Engebrecht et al., 1986).

Five gene lux A, B, C, D, E, respectively code for a subunit α and β ofluciferase, a fatty reductase, an acyltransferase and an acylproteinsynthase. Those enzymes enable oxidation of aldehyde into fatty acidwith production of photons. The aldehyde is then recycled by reductionof the fatty acid which has been formed.

The genes lux A, B, C, D, E, have been cloned without their regulon luxRand luxI forming thus an operon without the promoter and which is calledlux cassette (Schaw J. J. et al., 1988, "Transposon Tn4431 mutagenesisof Xanthonomas campestris pv campestris; characterization of anon-pathogenic mutant and cloning of a locus for pathogenicity" Mol.Plant-Microbe Interaction. 1:39-45).

According to another embodiment of the invention, the gene encodingresistance to a metal or encoding the catabolism of a xenobioticcompound originates from bacteria of the Alcaligenes eutrophus type.

The invention also relates to a fused gene, wherein:

the promoter and the gene encoding resistance is a promoter and a geneencoding resistance to zinc, obtained from pBR325 containing the czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 andsurrounding EcoRI fragment, digested with SalI, said promoter and geneencoding resistance is at the multiple cloning site of the plasmidpUCD615, said plasmid containing the lux operon of Vibrio fischeri.

The invention also relates to a fused gene, wherein:

the promoter and the gene encoding resistance is the promoter and geneencoding resistance to cobalt, obtained from pBR325 containing czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 digested withEcoRI-PstI, said promoter and gene encoding resistance is at themultiple cloning site of the plasmid pUCD615, said plasmid containingthe lux operon of Vibrio fischeri.

The invention also relates to a recombinant vector, particularly forcloning and/or expression, comprising a vector sequence, notably of thetype plasmid, cosmid or phage and a fused gene according to theinvention, in one of the non essential sites for its replication.

The invention also relates to a recombinant vector containing in one ofits non essential sites for its replication, necessary elements topromote, in a cellular host transcription and translation of the geneproducing a detectable signal and transcription, and possiblytranslation, of the gene responsible for the resistance to a metal orresponsible for the catabolism of a xenobiotic compound, and in additionto the inducible promoter possibly a signal sequence and/or anchoringsequence.

The invention also relates to a cellular host, notably E. coli,transformed by a recombinant vector according to the invention, orAlcaligenes eutrophus, transconjugated by a recombinant vector accordingto the invention, and comprising the regulation elements enabling theexpression of the gene producing a detectable signal and possibly theexpression of the gene encoding resistance to a metal or encoding thecatabolism of a xenobiotic compound.

An advantageous cellular host of the invention is E. coli transformed bya fused gene wherein

the promoter and the gene encoding resistance is a promoter and a geneencoding resistance to zinc, obtained from pBR325 containing the czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 andsurrounding EcoRI fragment, digested with SalI, said promoter and geneencoding resistance is at the multiple cloning site of the plasmidpUCD615, said plasmid containing the lux operon of Vibrio fischeri.

This cellular host forms a biosensor enabling to detect a range of about10 to about 65 ppm of zinc, and preferably as little as 0,1 ppm.

Another advantageous cellular host of the invention is E. colitransformed by a fused gene wherein:

the promoter and the gene encoding resistance is the promoter and geneencoding resistance to cobalt, obtained from pBR325 containing czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 digested withEcoRI-PstI, said promoter and gene encoding resistance is at themultiple cloning site of the plasmid pUCD615, said plasmid containingthe lux operon of Vibrio fischeri.

This cellular host forms a biosensor enabling to detect a range of about30 to about 120 ppm of cobalt, and preferably as little as 0,1 ppm.

Another advantageous cellular host of the invention is Alcaligeneseutrophus obtained by

the conjugation of Alcaligenes eutrophus and E. coli, E. coli containingthe vector pUCD623, itself containing a transposon Tn4431 which is Tn21transposon containing the tetracycline resistance and the lux operon ofVibrio fischeri without its own promoter,

the selection of the obtained transconjugants carried out ontetracycline plates,

the replication of the transconjugants on media with differentconcentrations of metals,

the detection of the light producing transconjugants being then carriedout.

Another advantageous cellular host of the invention is Alcaligeneseutrophus, obtained by conjugation of Alcaligenes eutrophus and of E.coli strain CM601, which gives AE714, transferred into A5.3 to giveAE859, which gives light expression in the presence of chromium.

This cellular host forms a biosensor enabling to detect a range of about20 to about 60 ppm of chromium, and preferably as little as 0,1 ppm.

Another advantageous cellular host of the invention is Alcaligeneseutrophus, obtained by conjugation of Alcaligenes eutrophus and of E.coli strain CM601, which gives AE453, transferred into A5.3 to giveAE891, which gives light expression in the presence of nickel.

This cellular host forms a biosensor enabling to detect a range of about5 to about 120 ppm of nickel, and preferably as little as 0,1 ppm.

Another advantageous cellular host of the invention is Alcaligeneseutrophus, obtained by conjugation of Alcaligenes eutrophus and of E.coli strain CM601, which gives AE866, which gives light expression inthe presence of copper.

This cellular host forms a biosensor enabling to detect a range of about1 to about 100 ppm of copper, and preferably as little as 0,1 ppm.

Another advantageous cellular host of the invention is Alcaligeneseutrophus, obtained by conjugation of Alcaligenes eutrophus and of E.coli strain CM601, which gives AE890, which gives light expression inthe presence of copper and cannot grow on minimal plates containinglead.

Another advantageous cellular host of the invention is Alcaligeneseutrophus, obtained by conjugation of Alcaligenes eutrophus and of E.coli strain CM601, which gives A5.23 or A5.24, which gives lightexpression in the presence of biphenyl compounds.

This cellular host forms a biosensor enabling to detect a range of about10 ppm, preferably as little as 1 ppb of biphenyl compounds, such as4-chloro-biphenyl.

The invention also relates to a process for in vitro preparing acellular host containing a fused gene comprising the following steps:

determination of the promoter and the gene encoding resistance to one orseveral metals or encoding the catabolism of one or several xenobioticcompounds and isolation of the corresponding nucleic acid fragment ofsaid promoter and gene, said promoter and gene comprising possibly amarker of the presence of the gene,

fusing said nucleic fragment with a gene producing a signal deleted fromits own promoter, said gene producing a signal comprising possibly amarker of the presence of the gene,

introducing the result of above-mentioned fusion into a cellular host,such as E. coli,

possibly selecting the cellular host with the marker(s) placed in amedium where the marker(s) can be detected,

detecting light producing cellular hosts placed in an appropriate mediumcontaining one or several metal(s) or a xenobiotic compound.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of zinc, wherein:

the promoter and the gene encoding resistance is a promoter and a geneencoding resistance to zinc, obtained from pBR325 containing the czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 andsurrounding EcoRI fragment, digested with SalI,

the result of the digestion is inserted into the plasmid pUCD615, at itsmultiple cloning site, and containing the lux operon of Vibrio fischeri,

the result of said insertion is cloned into E. coli,

a selection is carried out on ampicillin plates with variousconcentrations of zinc,

the detection of the light producing E. coli in the presence of zinc iscarried out.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of cobalt, wherein:

the promoter and the gene encoding resistance is the promoter and geneencoding resistance to cobalt, obtained from pBR325 containing czcfragment of pMOL30 from Alcaligenes eutrophus strain CH34 digested withEcoRI-PstI,

the result of the digestion is inserted into the plasmid pUCD615, as itsmultiple cloning sites, containing the lux operon of Vibrio fischeri,

the result of said insertion is cloned into E. coli,

the selection is carried out on ampicillin plates with variousconcentrations of cobalt,

the detection of the light producing E. coli in the presence of cobaltis carried out.

The invention also relates to a process for in vivo preparing a cellularhost containing a fused gene comprising the following steps:

conjugation, to obtain transconjugants, of a cellular host containing apromoter and a gene encoding the resistance to a metal or encoding thecatabolism of a xenobiotic compound and possibly a marker of thepresence of the gene, with another cellular host containing a transposoncontaining the gene emitting the detectable signal without its ownpromoter and possibly a marker of the presence of said gene,

recovery of the transconjugants,

possible selection of transconjugants with the marker(s) placed in amedium where the marker(s) can be detected,

possible application of transconjugants on media with differentconcentrations of metal or xenobiotics,

selection of transconjugants emitting light in the presence of a mediumcontaining a metal or a xenobiotic compound.

A preferred process for in vivo preparing a cellular host containingfused gene comprises the following steps:

the conjugation, to obtain transconjugants, of a cellular hostcontaining a promoter and a gene encoding the resistance to a metal orencoding the catabolism of a xenobiotic compound with another cellularhost containing a transposon containing the gene emitting the detectablesignal without its own promoter and a marker of the presence of saidgene, said marker being advantageously tetracycline,

the recovery of the cellular host containing said promoter and said geneencoding the resistance to a metal or encoding the catabolism of axenobiotic compound with elimination of the cellular host containingsaid transposon, by means of the marker and by means of a minimum mediumculture enabling the selection of only the cellular host containing saidpromoter and said gene encoding the resistance to a metal or encodingthe catabolism of a xenobiotic compound,

the application of the abovesaid transconjugants on media containing ornot the metal or xenobiotic compound, to select the transconjugantsemitting light only in the presence of a specific heavy metal or in thepresence of a specific xenobiotic compound.

For instance, when the cellular host containing said promoter and saidgene encoding the resistance to a heavy metal or to a xenobioticcompound is Alcaligenes eutrophus and when the cellular host containingthe transposon is E. coli CM601 (containing transposon Tn4431), theresistance to tetracycline enables to select on the one hand Alcaligeneseutrophus in which Tn4431 transposon has been inserted and on the otherhand strain CM601 which contains said transposon.

A minimum medium:

284 gluconate or

Schatz azelate,

on which CM601 cannot live because CM601 strains originates from HB101,autotrophic for leucine and proline, which enables to select onlyAlcaligenes eutrophus.

284 gluconate medium is as follows:

the basic composition of this culture medium is described in"Alcaligenes eutrophus CH34 is a facultative chemilithotroph withplasmid-bound resistance to heavy metals" M. Mergeay et al. (1985), J.Bacteriol. 162: 328-334; gluconate (0,2%) is used as carbon source).

Schatz azelate is described in Schatz A. et al. (1952) "Growth andhydrogenase activity of a new bacterium", Hydrogenomonas facilis. J.Bacteriol. 63:87-98; the carbon source which is used is azelate (0,2%)).

As the transposon can be inserted anywhere in the genome of Alcaligeneseutrophus, it is necessary to select the transconjugants in which thetransposon is inserted at a right place.

For this purpose, a film is deposited on the dishes containing thetransconjugants on said minimum medium, with or without a heavy metal ora xenobiotic compound. This technique enables to select the constitutiveconjugations (light emission independent on the presence of metal) fromthe non-specific inducible fusions (light emission in the presence ofone or several metals or under particular stress conditions) from thespecific inducible fusions (light emission in the presence of a specificmetal).

The detection of bacteria which have lost their resistance is alsocarried out on minimum medium in the presence of metals. This loss ordecrease in the resistance is due either because of an insertion of saidtransposon in the resistance gene or in its promoter or because of theloss of the plasmid carrying the resistance or a part of the plasmid.

More precisely, the in vivo fusion can be carried out as follows:

1) The strains CM601 and the strains presenting resistances with respectto metals and/or xenobiotic compounds are cultured in a liquid medium in5 ml of medium 869 for 16 h under stirring at 30° C. Medium 869 isequivalent to Luria-Broth medium: for 1 l of milli-q water:

10 q NaCl

5 g Bacto-Yeast extract

10 g Bacto-tryptone

adjust pH to 7.5 with sodium hydroxide.

2) 100 μl of culture are deposited on an agar dish (medium 869) in sucha way that the strains CM601 are deposited on one third of the dish, thestrains presenting resistance are deposited on a second third of thedish and both strains CM601 and the strains presenting resistance aredeposited on the last third of the dish.

3) After 2 days at 30° C., bacteria are recovered in the crossing areaand selected on the selective medium (Sz Azelate+Tet 20μg/ml=hist-medium, Tet+) on which only the recombinants grow (= bacteriawhich have inserted the Tn4431 transposon).

4) The recombinant are replicated on dishes containing different metals(in gluconate 284 medium) and the mutants which emit light in thepresence of specific metals are selected for further study.

The concentrations of the metal on the Petri dishes are the following:

284 gluconate+chromium 40 μg/ml

284 gluconate+nickel 2 mM

284 gluconate+cobalt 2 mM

284 gluconate+zinc 2 mM

284 gluconate+cadmium 0.8 mM

284 gluconate+lead 0.3 mM

284 gluconate+copper 0.8 mM

A preferred cellular host is Alcaligenes eutrophus type are interestingfor the following reasons:

they present a high ability of specific resistance expression to ametal,

these mechanisms are inducible (Siddigui et al., 1988, Inductible andconstitutive expression of pMOL 28-encoded nickel resistance inAlcaligenes eutrophus N9A. J. Bacteriol. 170, 4188-4193; Nies et al.,1989, Plasmid-determined inductible efflux is responsible for resistanceto cadmium, zinc, cobalt and nickel in Alcaligenes eutrophus. J.Bacteriol. 171, 896-900; Senfuss et al., 1989, Plasmid PMOL 28 encodedresistance to nickel is due to a specific efflux. FEMS Microbiol. Lett.55, 295-298);

it is a good recipient for exogenous genes in heterospecificconjugations (Lejeune et al., 1983, Chromosomal transfer and R-primeplasmid formation mediated by plasmid pULB113 (RP4::Mini-Mu) inAlcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2. J.Bacteriol. 155, 1015-1026).

The invention also relates to a process wherein the cellular hostcontaining a promoter and a gene encoding the resistance to a metal isAlcaligenes eutrophus and the cellular host containing a transposon isE. coli containing the vector pUCD623, itself containing a transposonTn4431 which is Tn21 transposon containing the tetracycline resistanceand the lux operon of Vibrio fischeri without its own promoter,

the selection of the obtained transconjugants is carried out ontetracycline plates,

the transconjugants are replicated on media with differentconcentrations of metals,

the detection of the light producing transconjugants is carried out.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of chromium, wherein the cellular hostcontaining a promoter and a gene resistant to a metal is Alcaligeneseutrophus SV661 and the cellular host containing the transposon is E.coli strain CM601, which gives AE714, transferred into A5.3 to giveAE859, which gives light expression in the presence of chromium.

The selection of the chromium biosensor is advantageously carried out(besides the marker and minimum medium) in the presence of zinc whichenables to select the transconjugants which have not inserted saidtransposon in the zinc resistance gene.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of nickel, wherein the cellular hostcontaining a promoter and a gene resistant to a metal is Alcaligeneseutrophus AE631 and the cellular host containing the transposon is E.coli strain CM601, which gives AE453, transferred into A5.3 to giveAE891, which gives light expression in the presence of nickel.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of copper, wherein the cellular hostcontaining a promoter and a gene resistant to a metal is Alcaligeneseutrophus DS185 and the cellular host containing the transposon is E.coli strain CM601, which gives AE866, which gives light expression inthe presence of copper.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of copper, wherein the cellular hostcontaining a promoter and a gene resistant to a metal is Alcaligeneseutrophus DS310, and the cellular host containing the transposon is E.coli strain CM601, which gives AE890, which gives light expression inthe presence of copper and cannot grow on minimal plates containinglead.

The invention also relates to a process for preparing a cellular hostemitting light in the presence of a biphenyl compound, wherein thecellular host containing a promoter and a gene encoding the catabolismof biphenyl compounds is Alcaligenes eutrophus A5 and the cellular hostcontaining the transposon is E. coli strain CM601, which gives A5.23 orA5.24, which gives light expression in the presence of biphenylcompounds.

The invention also relates to E. coli liable to be transformed accordingto the process of the invention.

The invention also relates to Alcaligenes eutrophus liable to betransconjugated according to the process of the invention.

The invention also relates to a process for detecting, on a solidmedium, a metal or a xenobiotic compound, preferably in a concentrationrange of about 1 to about 120 ppm, comprising:

the use of a solid support, such as an agar disc containing anappropriate solid medium for a cellular host of the invention,

the application, on said agar disc, of a cellular host of the inventioncontained in a liquid medium,

placing a radiographic film under the above-mentioned agar disc,

detecting the bioluminescence by comparison on the film of theblackening of the film.

The invention also relates to a process for detecting, in a liquidmedium, a metal or a xenobiotic compound, preferably in a concentrationrange of about 1 to about 120 ppm, comprising:

placing cellular hosts of the invention which have been lyophilyzed andimmobilized on a solid support, into a liquid medium,

introducing a sample of said liquid culture medium, containing cellularhosts of the invention, in a sample taken from a liquid medium, such aswater, in which the presence of a metal or of a xenobiotic compound isto be detected,

detecting the signal, for instance, the light generated by the presenceof said metal or the presence of said xenobiotic compound, by detectingmeans such as a luminometer.

The invention also relates to a process for detecting in a liquid mediuma metal or a xenobiotic compound, preferably in a concentration range ofabout 1 to about 120 ppm, comprising:

introducing a cellular host of the invention contained in a liquidmedium, into a sample taken from a liquid medium, such as water,

detecting the signal, for instance, the light generated by the presenceof said metal or the presence of said xenobiotic compound by detectingmeans such as a luminometer.

The invention also relates to a kit for detecting a metal or axenobiotic compound in a concentration range as little as 0,1 ppm formetals and as little as 1 ppb for xenobiotics, comprising:

a cellular host of the invention,

detection means, for instance to detect the light generated by thepresence of said metal or xenobiotic compound, such as a luminometer.

By way of example:

1) a preculture is obtained by inoculation of an isolated colony of acellular host of the invention in a rich liquid medium such as 869,preferably containing 20 μg/ml of tetracycline, to select only thecellular hosts in which the transposon has been inserted;

2) the culture is diluted, for instance 20 times, in the liquid samplecontaining the metal or xenobiotic to be determined in a final volume ofabout 0,5 ml;

3) bioluminescence is measured, for instance with a luminometer.

COMMENTS ON THE FIGURES

FIG. 1 represents bioluminescence by zinc (in the form of Zn²⁺) inducedstrain CM685 expressed in bioluminescence units (cpm) plotted againstthe time (in hours).

Strain CM685 was cultured overnight in liquid medium 869. Aliquots of 10μl were applied on standardized agar discs with or without 1 mM zinc(see materials and methods) which were transferred into sterile vials ofa scintillation counter. Bioluminescence was monitored automaticallyevery hour.

The results represent average values of triplicate samples and theassociated standard error of the mean (S.E.M.). The curve comprising thetriangles correspond to samples containing 1 mM Zn²⁺. The curvecomprising the circles correspond to control samples.

FIG. 2 represents the signal/noise ratio plotted against time (in hours)for bioluminescence of zinc (in the form of Zn²⁺) induced strain CM685on agar discs.

Strain CM685 was cultured overnight in liquid medium 869. Aliquots of 10μl were applied on standardized agar discs which contained 1 mM zinc.The measured bioluminescence signal was divided by the signal ofparallel vials containing the same amount of bacteria growing on agardiscs without zinc.

FIG. 3 represents the cobalt (in the form of Co⁺⁺) inducedbioluminescence expressed in mV/versus the time (in hours) on solidagar.

Strain CM781 was cultured overnight in liquid medium 869. After 20 folddilution, 10 μl aliquots were. evenly distributed over the surface ofpunched out mini agar discs containing increasing concentrations ofCo⁺⁺.

Bioluminescence was monitored automatically every 30 min after transferof the agar discs into the bottom of sterile luminometer tubes. The gainof the photomultiplier is stabilized automatically in this instrument,facilitating measurements over long periods.

The results are the average from 5 duplicate samples.

The curve comprising "+" correspond to samples containing no Co⁺⁺.

The curve comprising triangles correspond to samples containing 0,2 mMCo⁺⁺.

The curve comprising circles correspond to samples containing 0,5 mMCo⁺⁺.

The curve comprising "+" correspond to samples containing 1,0 mM Co⁺⁺.

FIG. 4 represents chromium (in the form of chromate) inducedbioluminescence on agar (expressed in relative light units) containingminimal medium versus the amount of chromium added.

Strain AE859 was grown in minimal liquid medium 284+gluconate during 70h. Aliquots of the undiluted culture were transferred on standardizedagar discs as described in materials and methods.

The total light output during 3 days growth in the luminometer wascalculated for each group and corrected for differences in growth, asmeasured by turbidimetry. The resulting relative light output is plottedagainst the chromium concentrations used.

FIG. 5 represents the signal/noise ratio of AE859 grown on agar 869versus the amount of chromium (in the form of chromate) added (in mM).

Strain AE859 was grown during 23 h in liquid medium 869. Undilutedaliquots of 10 μl were transferred on standardized discs of agarcontaining growth medium 869. The signal/noise ratio is calculated asindicated in FIG. 2 except for the length of the growth in theluminometer which was 2 days. No light was produced after more than 20hours. The results are corrected for small differences in growth.

FIG. 6 represents the bioluminescence (in mV)of AE866 versus the copper(in the form of Cu²⁺) concentrations (in ppm).

Strain AE866 was grown during 16 h in liquid medium 869. Undilutedaliquots of 10 μl were transferred as described above. Each valuerepresented the maximum mean value of 15 aliquots at different copperconcentrations.

FIG. 7 represents bioluminescence (expressed in cpm) versus the time, ofstrains inducing light in the presence of chlorinated compounds.

Strain A5-24 was grown on agar discs containing minimal medium284+gluconate during the time indicated. Small cristals of biphenyl or4-chlorobiphenyl were placed at the bottom of the scintillation vialwithout direct contact with the agar discs. Transformer oil (askarel)(10 microliters) was also deposited next to the agar discs in such a waythat only volatile components could reach the bacterial growth.

The curve with circles corresponds to control samples.

The curve with black triangles pointing downward corresponds to samplescontaining biphenyl.

The curve with black squares corresponds to samples containing4-chlorobiphenyl.

The curve with black triangles pointing upward corresponds to samplescontaining transformer oil.

FIG. 8 represents an enhanced bioluminescence of strain A5.23 afterpreadaptation to biphenyl.

The curve containing black triangles pointing downward corresponds tothe bioluminescence in the presence of biphenyl of strain A5.23 afterpreadaptation to biphenyl.

The curve containing circles corresponds to the bioluminescence in theabsence of biphenyl of strain A5.23 after preadaptation to biphenyl.

Strain A5-23 grown during 3 days on minimal agar 284+gluconate in thepresence of volatile biphenyl cristals, was harvested, resuspended in asmall volume (50 μl) liquid medium 284+gluconate without inducer andtransferred in 8 μl aliquots to fresh agar discs with or withoutbiphenyl cristals. Bioluminescence measurements were started immediatelythereafter.

FIG. 9 represents the cartography of plasmid pBR325 (5.6 kb).

FIG. 10 represents the cartography of plasmid pMOL149 (20.7 kb).

FIG. 11 represents a DNA sequence identified by SEQ ID NO:1 (top strand)and by SEQ ID NO:2 (bottom strand) of the EcoRI-SalI fragment of aE23 ofpMOL 149 (see FIG. 10), said DNA sequence enabling the induction of luxgenes by zinc. The ORF are represented in darker characters.

FIG. 12 represents the specificity of a copper biosensor (AE984 inmedium 869 containing tetracycline Tc!) compared to Cu, Cd, Co, Zn, Pb,Ni and biphenyl. Total bioluminescence after 24 h is expressed in mV.

FIG. 13 represents the specificity of the same copper biosensor as inFIG. 12, compared to Cu, Cd, Zn, Ni, Co, Cr, Mn, Ag, Hg and Tl (atdifferent concentrations). Bioluminescence is measured during 10 secondsand expressed in relative light units (RLU).

The metals have been used at the following concentrations: ##STR1##

In other words:

the black pattern corresponds to a concentration of 0.01 mM of metal,

the fine cross hatching pattern corresponds to a concentration of 0.1 mMof metal,

the diagonal lines pattern corresponds to a concentration of 0.2 mM ofmetal,

the coarse cross hatching pattern corresponds to a concentration of 0.4mM of metal.

MATERIALS AND METHODS

In the following example, the cellular hosts emitting light in thepresence of a metal or in the presence of a xenobiotic compound will benamed "biosensors".

Bacterial strains and plasmids

The metal resistance genes, used for the gene fusions, are isolated fromAlcaligenes eutrophus CH34 ("Alcaligenes eutrophus CH34 is a facultativechemilithotroph with plasmid-bound resistance to heavy metals" M.Mergeay et al. (1985), J. Bacteriol. 162: 328-334) or related strains(Diels L. et al., 1990, "DNA probe-mediated detection of resistantbacteria from soils highly polluted by heavy metals" Appl. Environ.Microbiol. 56:1485-1491). On the other hand, the biphenyl degradinggenes come from another Alcaligenes eutrophus strain A5 (Shields M. S.et al., 1985, "Plasmid-mediated mineralization of 4-chlorobiphenyl", J.Bacteriol. 163:882-889).

Construction of New Fusions By in Vitro Cloning

a) Construction of a zinc biosensor:

A zinc biosensor was constructed by cloning in E. coli (S17/1). A SalIfragment (3.5 kb) from pMOL149 (hereafter described) (pBR325 with theczc fragment of pMOL30 from CH34 and its surrounding EcoRI fragment) wasinserted in the promoter expressing vector pUCD615 (said vector beingcontained in a strain of E. coli, CM600 deposited at the C.N.C.M.,Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991,under n° I-1050). Plasmid pBR325 comprises a complete copy of pBR322(ATCC No. 31344; U.S. Pat. No. 4,342,832 and No. 4,366,246) (I-A-iv-I)opened at the EcoRI site and a 1.2 kb HaeII fragment containing the cmlgene. Plasmid pBR325 has been certified by the NIH Recombinant DNAAdvisory Committee as an EK2 vector (Recombinant DNA Technical Bulletin,NIH5 (1982)). The nucleotide sequences of pBR325 is known. The digestionwith SalI in the multiple cloning site of pUCD615 and in pMOL149 aredone according to Maniatis et al. (1982), p. 104-105. After thedephosphorylation (Maniatis et al., 1982, p. 133-134) of pUCD615, theligation (Maniatis et al., 1982, p. 125-126) with the SalI fragments wascarried out. Selection was done on ampicillin plates with 0,5 mM ZnCl₂.Light producing colonies were selected with autoradiography and withPolaroid photography.

This biosensor is hereafter designated by CM685.

The detailed protocol is given hereafter.

1. Curing of CH34 and creation of AE128:

An erlenmeyer flask (50 ml) containing 5 ml of 284-gluconate medium withmitomycin C (4 μg/ml) was shaken at 30° C. during 5 days. Cells from theflask were harvested, washed, diluted and spread on agar platescontaining 284-gluconate with 2 mM NiCl₂. Plasmid-deficient mutantsoccurred at a frequency of 10⁻³ to 10⁻⁵ per mitomycin C treated cell. Nisensitive cells contained only pMOL30 and this was evaluated by agarose(0.8%) gel electrophoresis. This resulting strain was registered asAE128.

2. Isolation of pMOL30 from AE128:

An overnight culture of AE128 in medium M3 (Nutrient Broth (Difco) 8g/l) (30 ml) was centrifuged during 10 minutes at 4000 rpm in 6 tubes of5 ml. Each pellet was suspended in 1 ml E buffer (0.04M Tris-acetate pH7,9; 0,002M EDTA). Afterwards, 2 ml lysis buffer (3% SDS; 0.05M Tris-OHpH 12.55) was added and incubated is glasstubes at 65° C. during 60minutes.

Afterwards, 400 μl 5M NaCl and 6 ml phenol/CHCl₃ were added and mixedfollowed by a centrifugation of 10 minutes. Then the tubes wereincubated at 4° C. during 1 hour. The bottom phase was eliminated andthe top phase centrifuged again (10 minutes).

The top phase was casted in a siliconised glass tube. 30 μl 10% aceticacid was added and 6 ml diethyl ether and agitated. After centrifugationand removal of the ether layer, the tubes were incubated at 65° C.during 10 minutes to remove the traces of ether. The DNA wasprecipitated with 50 μl 5 mM NaCl and 2 ml ethanol. After a 2 hourincubation at -15° C. the tubes were centrifuged during 15 minutes. Allthe six pellets were dissolved in 2.0 ml water, 200 μl 5 M NaCl and 4.4ml ethanol added. After a 2 hour incubation at -15° C. the tubes werecentrifuged and the pellet dried and dissolved in 200 μl H₂ O.

3. Digestion of pMOL30 with EcoRI:

To 20 μl of pMOL30 2.5 μl of 10×EcoRI buffer, 4 μl RNase solution and 1μl EcoRI (50 U/μl) were added. After incubation at 37° C. during 2hours, the DNA was treated with a phenol extraction and precipitatedwith ethanol. The DNA pellet was dissolved in 50 μl TE buffer.

4. Digestion of pBR325 with EcoRI:

To 20 μl of pBR325 (cf. FIG. 9) (2 μg/10 μl) 2.5 μl of 10×EcoRI buffer,4 μl RNase solution and 0.5 μl EcoRI (50 U/μl) were added. Afterincubation at 37° C. during 2 hours, the DNA was treated with a phenolextraction and precipitated with EtOH.

5. Dephosphorylation of EcoRI digests of pBR325:

To a pellet 35 μl 10 mM Tris-HCl pH 8 buffer and 4 μl of CIP buffer(0.5M Tris-HCl pH 9.0; 10 mM MgCl₂ ; 1 mM ZbCl₂ ; 10 mM spermidine+2 μlCIP (0.5 U) were added and incubated at 37° C. during 30 minutes.Afterwards, again 2 μl C/P was added for a second incubation of 30minutes.

50 μl water and 10 μl 10×STE (100 mM Tris-HCl pH 8; l mM NaCl; 10 mMEDTA and 1 μl 20% SDS were added and incubated at 68° C. during 15minutes. One phenol/CHCl₃ and one CHCl₃ extraction were done followed byan ethanol precipitation with 10 μl 5M NaCl and 330 μl EtOH(precipitation at -70° C. during 15 minutes).

6. Ligation of pBR325 EcoRI with EcoRI digests of pMOL30:

To the pellet of (5) 40 μl H₂ O and 5 μl ligation buffer (0.5 m Tris pH7,4; 0.1 m MgCl₂ ; 0.1M dithiotreitol; 10 mM spermidine; 10 mM ATP; 1mg/ml BSA and 12 μl of (3) were added and ligation was done with 4 μlligase (10 to 20 U).

An overnight incubation of this ligation mixture was done at 12° C.Afterwards, 50 μl TE buffer (10 mM Tris-HCl pH 7,4; 1 mM EDTA) was addedand after a phenol/CHCl₃ and CHCl₃ extraction, the DNA was precipitatedwith 10 μl 5M NaCl and 330 μl EtOH.

7. Transformation of HB101:

Transformation of E. coli HB101 was done according to the CaCl₂ methodof Maniatis et al. Selection was done for Tet^(R), Amp^(R) and Cm^(S)clones.

Clone CM485 which contained pMOL149 (cf. FIG. 10) being pBR325 with aE8,aE23, aE38 and aE39 of pMOL30. pMOL149 was isolated according toBirnboin and Dolly (Nucl. Acid. Res. 7:1513).

8. SalI digestion of pMOL149:

pMOL149 was digested in the same way as explained in (3) 1 μl SalIenzyme (50 U) and 2.5 μl SalI digestion buffer was used.

9. SalI digestion of pUCD615:

pUCD615 (isolated from E. coli CM600 deposited at the C.N.C.M., InstitutPasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, under n°I-1050 according to Birnboin above-mentioned) was digested in the sameway as pMOL149 (8).

10. Dephosphorylation of pUCD615 SalI:

The dephosphorylation of pUCD615 SalI was done in the same way asexplained in (5).

11. Ligation of pMOL149 SalI with pUCD615 SalI:

The ligation between pMOL149 SalI and pUCD615 SalI was done according to(6).

12. Transformation of the ligate in S17/1:

The ligate was transformed into S17/1 according to Maniatis et al.(p250). Selection was done on Amp^(R) transformants.

13. Selection of phenotype lux⁺ with 0.5 mM Zn:

Amp^(R) transformants were replicated on Petri dishes with LB broth+50μg Amp with addition of 0, 0.1, 0.2, 0.5, 1.0 mM ZnCl₂. These disheswere put on X-ray films during incubation in cardboard boxes. Coloniesgiving more and more light at increasing Zn concentrations were selectedand purified and their phenotype further analysed in a luminometerexperiment.

b) Construction of a cobalt biosensor:

A cobalt biosensor was created in the same way as the above describedzinc biosensor by inserting an EcoRI-PstI fragment (<0.1 kb) frompMOL149 in pUCD615. The PstI site was made blunt end (Maniatis et al.(1982), p. 394-395) and afterwards a phosphorylated EcoRI linker wasattached (Maniatis et al. (1982), p.396-397) to that site. The linkerwas digested with EcoRI (Maniatis et al. (1982), p. 104-105) and thefragment was ligated (Maniatis et al. (1982), p.125-126) to pUCD615.Selection was done on ampicillin plates with 0.5 mM CoCl₂. Lightproducing colonies were selected with autoradiography.

This biosensor is hereafter designated by CM781.

Construction of New Fusions By In Vivo Cloning

Different Alcaligenes strains were conjugated with the E. coli strainCM601 (deposited at the C.N.C.M., Institut Pasteur, 28 rue du Dr Roux,75015 Paris, on Feb. 28, 1991, under n° I-1051) bearing the suicidevector pUCD623 with the lux transposon Tn4431 (without its ownpromoter). After conjugation, transconjugants were selected ontetracycline plates. Afterwards transconjugants were replicated on mediawith different concentrations of heavy metals. In this way differentbiosensors could be constructed.

a) Construction of a chromium biosensor:

A chromium biosensor could be obtained by conjugation of the chromiumresistant Alcaligenes eutrophus SV661 strain (deposited at the C.N.C.M.,Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28, 1991,under n° I-1046) (Diels L. et al., 1990, "DNA probe-mediated detectionof resistant bacteria from soils highly polluted by heavy metals" Appl.Environ. Microbiol. 56:1485-1491) with CM601. Selection was done onmineral medium (Schlegel H. G. et al., 1961, "Ein submersverfahren zurkultur wasserstoffoxidierender bakterien: wachstums physiologischeuntersuchungen" Arch. Mikrobiol. 38:209-222) with 20 mg/l tetracyclineand 2 mM zinc. The obtained transconjugants were transferred on mineralplates with 1 mM CrO₄ ⁻. Light emitting colonies were selected byautoradiography.

This biosensor is hereafter designated by AE714.

The construction of the invention contained in AE714 was transferred inA5.3 to give a stable strain AE859.

b) Construction of a nickel biosensor:

A nickel biosensor was obtained after mating between AE453 and CM601(AE453 has been deposited at the C.N.C.M., Institut Pasteur, 28 rue duDr Roux, 75015 Paris, on Feb. 28, 1991, under n° I-1049). Afterselection on tetracycline the transconjugants were selected on mineralmedium with 0,5 mMNiCl₂.

This biosensor is hereafter designated by AE631.

The construction of the invention contained in AE631 was transferred inA5.3 to give a stable strain AE891.

c) Construction of a copper biosensor:

Copper biosensor were obtained after mating between DS185 or DS310bearing each pMOL90, 85 and 80 (Diels L. et al., 1990, "DNAprobe-mediated detection of resistant bacteria from soils highlypolluted by heavy metals" Appl. Environ. Microbiol. 56:1485-1491) andCM601.

DS185 has been deposited at the C.N.C.M., Institut Pasteur, 28 rue du DrRoux, 75015 Paris, on Feb. 28, 1991, under n° I-1048.

DS310 was obtained from DS185 as follows:

A 5 ml culture of DS185 in 284 gluconate medium with SDS (0.01%) wasshaken in an erlenmeyer flask (50 ml) at 30° C. during 4 days. Cellsfrom the flask were harvested, washed, diluted and spread on agar platescontaining 284 gluconate with 2 mM Zn. Plasmid analysis was performedaccording to Kado C. I. et al. (1981), "Rapid procedure for detectionand isolation of large and small plasmids" J. Bacteriol. 145:1365-1373.DS310 was obtained by this way and had lost the pMOL80 plasmid (4 kb).

From the mating between DS185 and CM601, 200 colonies were tested andfrom the mating between DS310 and CM601, 200 colonies were also tested.

The selection was realized as described above on minimal plates with 0,8mM copper as inductive agent and tetracycline.

These biosensors obtained respectively with DS185 and DS310 arehereafter designated by AE866 and AE890.

d) Construction of biphenyl biosensor:

Two biphenyl biosensors were obtained after mating between A5 and CM601.After selection on tetracycline the transconjugants were screened forlight induction on minimal plates with biphenyl as inductive agent.

These biosensors are hereafter designated by A5.23 and A5.24.

Conjugation With A5.3

The in vivo made constructions in Alcaligenes eutrophus var.metallotolerane contained rather unstable Tn4431 insertions. Thereforethe plasmids were transferred to A5.3. The strain A5.3 is a rifampicinmutant of the biphenyl degrading strain A5 (A5 has been deposited at theC.N.C.M., Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28,1991, under n° I-1047). This mutant is obtained by spreading A5 on agarplates containing Luria Broth medium with 100 μg/ml rifampicin.Resistant colonies are selected. After conjugation, selection was doneon minimal plates containing tetracycline, rifampicin. The obtainedtransconjugants were tested for their resistance to chromium (biosensordesignated by AE859) or nickel (biosensor designated by AE891)respectively and for their light expression on these metals.

Measurement of Bioluminescence

Luminescence was quantitated with a scintillation counter (PackardTri-Carb model 2425) set in the chemiluminescence detection mode or witha luminometer (Bio-Orbit model 1251). In the former instrument, thebioluminescent activity was reported in cpm whereas in the latterinstrument, bioluminescence is expressed in mV.

Induction experiments were performed on cultures of mutant strains insterile vials cycled continuously for the duration of the experiment inthe bioluminescence counter.

To measure optical density from liquid cultures, samples were removedfrom larger parallel cultures.

To measure bacterial growth at the end of cultures grown on calibratedagar discs (9 mm .o slashed., 3,5 mm thick), the bacteria were dislodgedfrom the agar by vigorous shaking during 1 h in 2 ml MgSO₄ 10 mM inclosed tubes.

The turbidity of the supernatant was read at 630 nm using a Perkin Elmerspectrophotometer model lambda 3.

Experiments with fusion strains were performed at or below 30° C. sinceluciferase is inactivated at higher temperatures. Luminescence, wherereported in relative light units, is normalized through division by themeasured turbidity of the cultures.

Semi-quantitative comparative measurement of bioluminescence by severalstrains on different media was performed as follows:

Petri-dishes containing the appropriate solid medium were calibrated byweight after 30 minutes drying at 30° C. The net weight of the agar waschosen so as to obtain an average thickness of the agar of 3,5±0,1 mm inthe central portion of the plate.

Using a sterile cork-bore with an internal diameter of 9 mm, agar discswere punched out from the central portion of the agar and transferredwith a small spatula to sterile, empty Petri-dishes.

A liquid preculture, grown during the appropriate time was applied,diluted or not, in 10 microliter aliquots on each 9 mm agar minidisc.Care was taken during pipetting to disperse the culture evenly over thewhole surface of the minidiscs. Triplicate (or more) samples are usedfor each group.

The Petri dishes are fixed in dark plastic (4 mm thick) plates, providedwith 6 circular cutouts wherein the dishes fit snugly.

A radiographic film (Kodak Ortho G) is placed under the Petri-disheswhich are exposed in triple cardboard boxes during several hours ordays, at the optimal temperature.

At the end of the exposure, the film is developed and the intensity ofbioluminescence is judged by comparison of the blackening of the filmunder the minidiscs. If desired, corrections for differences in overallgrowth can be made by turbidimetry of the resuspended bacteria (v.s.).

This method gives a cumulative result of total light output during agiven period but does not allow easily to follow the time-dependentlight emission which is better quantitated using repeated measurementsin a programmable luminometer. When more quantitative results over agiven time segment were desired for bacteria, grown on solid agar,calibrated agar minidiscs were transferred to vials, appropriate for thebioluminescence counter of choice and measurements were performed atregular intervals on triplicate samples in sterile conditions.

Results A. Light-Emitting Bacteria, Inducible By Heavy Metals

1) CM685: zinc biosensor

Different SalI fragments could be inserted in pUCD615. One of themcontaining plasmid pLD13 produced light on zinc plates. Another strain,bearing plasmid pLD10 produced also light on zinc plates but seemed tobe extremely sensitive to zinc ions. Plasmid pLD13 contains a 3.5 kbSalI fragment overlapping the left site of the czc operon of pMOL30 fromCH34 (ATCC 43123).

The bioluminescence, induced by 1 mM zinc⁺⁺ in solid growth medium isdepicted in FIG. 1. During the first 7 hours, the toxicity of 1 mMzinc⁺⁺ is sufficiently high in this non-resistant E. coli strain toretard growth and decrease the light output in comparison with thecontrol group.

After the induction period of about 8 h, the light output increasesdramatically and reaches a level, at least 10 times higher than that ofthe control group.

This enhanced bioluminescence is not due to better growth of bacteria onthe zinc-containing agar because earlier experiments showed the contrary(data not shown). If the bioluminescence of the control group withoutzinc is taken as noise, a signal/noise ratio can be determined for eachtime period during 25 h of measurement. From FIG. 2, it follows thatthis ratio is very time-dependent in a complex way. The highestsignal/noise ratio (86) is obtained after 21 hours.

When tested in liquid medium, this strain demonstrates only a marginallyincreased luminescence (+21% n=12) in the presence of 0,5 mM zinc during24 hours at 30° C. with intermittent agitation in the luminometer.

The zinc promoter is characterized by the fact that it comprises afragment of at least 20 contiguous base pairs of the DNA represented onFIG. 11, said fragment enabling the induction of lux genes by zinc.

2) CM781: cobalt biosensor:

From the several clones, obtained after EcoRI-PstI fragment insertion,one clone, emitting light on cobalt plates, could be obtained. Theintroduced EcoRI-PstI fragment is a very small one (<0.1 kb) and coulduntil now not be identified in a clear way.

Cobalt being more toxic than zinc, different concentrations of thisheavy metal were tested in solid nutrient agar.

Increased Co⁺⁺ concentrations give rise to increased overall lightoutput over a period of 48 hours. From FIG. 3 it is also obvious thatthe maximal bioluminescence is reached later when the Co⁺⁺ concentrationincreases.

In this strain, bioluminescence in liquid cultures decreases during a 24h measuring period in the presence of increasing Co⁺⁺ concentrations(data not shown). However, recent experiments indicate that growth inthe luminometer-vials is very slow and after 35 hours at 28,0° C. withintermittent agitation every 10 minutes, a considerable increase inbioluminescence is observed in the controls.

3) AE714: chromium biosensor:

The conjugation between SV661 and CM601 resulted among others in a Ni⁻,Cr⁺ AE714 mutant obtained by introduction of Tn4431 in pMOL28.661. Thetransposon Tn4431 was not very stable in Alcaligenes eutrophus var.metallotolerans and for that reason the plasmid pMOL28.661::Tn4431 wastransferred to strain A5.3, a rifampicin resistant biphenyl degradingstrain. This resulted in strain AE859 with a stable light expression onchromium ions.

In this strain also, the growth of the bacteria on the chromiumcontaining agar was markedly inferior to that of the controls, as judgedvisually. At higher chromium concentrations, growth is very poor andlight production faint (date not shown).

4) AE859: chromium biosensor:

This construct is more stable than strain AE714. When grown on minimalmedium 284 glu during 3 days, the presence of chromium ions produces alinear increase in light output until 0.2 mM (FIG. 4).

Growth and light production are faster when this strain is grown on agarcontaining a rich nutrient broth 869 but the signal/noise ratio reachesonly a value of 2,06±0,03 at the highest chromium concentration tested(0.5 mM)(FIG. 5). This is due to the high background bioluminescence ofthe control group where the lux genes are not completely "silent" in theabsence of added chromium. One possibility to explain this background isthe presence of cryptic promoter sequences, unrelated to the heavy metalinducible promoter present in this mutant.

5) AE891: nickel biosensor:

The resulting strain from the conjugation of AE453 with CM601 was AE631containing an insertion of Tn4431 in the ZinB gene resulting in a Zin⁻strain. Also this strain was unstable and therefore pMOL55::Tn4431 wasagain transferred to A5.3 resulting in AE891 presenting a stable lightemitting construction on nickel plates.

In the presence of at least 0,5 mM Ni⁺⁺ in minimal nutrient agar (284glu) an increased bioluminescence is observed after 2 days of growth.This increase is still manifest at 1 and 2 mM Ni⁺⁺ where toxicitybecomes limiting for adequate growth. The maximal signal/noise ratio(8,2±0,5) was reached in the presence of 1 mM Ni⁺⁺. The induction periodfor increased luminescence is at least 10 h at 30° C. in the presence of0,5 mM Ni⁺⁺. At higher Ni⁺⁺ concentrations the induction periodincreases considerably.

6) AE866 and AE890: copper biosensors:

The conjugation DS185 with CM601 gave AE866 by insertion of Tn4431 inpMOL85. The insertion of Tn4431 is located on pMOL85. The transconjugantAE890 results from the conjugation between DS185 and CM601. AE890 issensitive for lead. A linear light response was observed for AE866between 1 and 100 ppm on solid agar containing a rich nutrient broth 869(FIG. 6). The light emission peak is obtained 9 to 10 h after inductionand the detection limit with a signal/noise ratio of 2 was about 10 ppmcopper. Above 100 ppm, light response was not more linear because of thetoxicity of copper on the bacterial growth.

7) AE984: copper biosensor:

The strain AE984 is a derivative of strain AE866 which has lostspontaneously pMOL85 and which contains an insertion of Tn4431 in pMOL90(pMOL90::Tn4431).

FIG. 12 represents the specificity of AE984 with respect to copper. Thebackground noise is obtained with the control sample. The specificityhas been determined in the following conditions:

The strain AE984 was grown overnight at 30° C. The next day, dilutionswere made with an optical density of 0.1.

Every test was done 3 times. Metal solutions were added to differenttubes, to obtain final concentrations of:

control

0.5 mM Cu

0.25 mM Cd

0.25 mM Co

0.5 mM Zn

0.25 mM Pb

0.25 mM Ni

100 ppm Biphenyl.

Measurements were done in a Luminometer with 49 cycles every 30 minutes.Light is expressed in mV and the highest obtained values are presented.

When the tubes tested contain one of the following elements: Cd, Co, Zn,Pb, Ni or chlorinated biphenyl, no significant bioluminescence isobserved.

7bis) AE984: copper biosensor:

In a second experiment strain AE984 was grown overnight at 30° C. Thenext day, dilutions were made to an optical density of 0.1.

Metal solutions were added to different tubes, to obtain finalconcentrations of 0.01; 0.1; 0.2 and 0.4 mM of the following metals:

control,

Cu,

Cd,

Zn,

Ni,

Co,

Cr,

Mn,

Ag,

Hg,

Tl.

Measurements were done in a Lumac luminometer after 18 hours ofincubation at 21° C. Light was expressed in Relative Light Units(R.L.U.).

The results of these experiments are represented on FIG. 13.

8) Construction of thallium biosensors: AE1053, AE1060, AE1101:

A Tl sensor was constructed by conjugation between E. coli CM601 and A.eutrophus AE126.

The strain AE126 can be obtained by curing of CH34 with mitomycine C (5μg/ml, 2 days incubation) and contains only plasmid pMOL28. This strainis sensitive to Zn (tested with ZnCl₂) and resistant with respect to Ni(tested with NiCl₂).

After conjugation, selection of transconjugants was done on minimalmedium plates with gluconate as carbon source and with 20 μg/ml oftetracycline to select AE126 strains bearing the transposon. Afterwards,the transconjugants were tested on rich media (nutrient broth) with orwithout Tl and incubated with an autoradiography film on top of thePetri dishes. Strains inducing light in the presence of Tl wereselected. The best strains were named AE1053, AE1060 and they werehighly specific for Tl. No light induction was obtained by Co, Cs, orCd. A very small induction could be obtained by Ni and Hg. Light wasinduced by insoluble (e.g. Tl₂ S) and soluble (e.g. TlNO₃) compounds.

The light transposon Tn4431 was inserted in A. eutrophus chromosome ascould be shown by hybridization of the transposon with A. eutrophuschromosome and plasmid DNA.

Strain AE1053 was afterwards conjugated with A5.3 (a rifampycinresistant A5 strain) and selection was done on minimal plates withrifampycin (100 μg/ml) and tetracycline (20 μg/ml). The resulting strainwas AE1101 and displayed also light in function of increasing Tlconcentrations.

Light was induced in the three strains AE1053, AE1060 and AE1101 by Tlconcentrations between 0.005 mM and 0.02 mM for Tl₂ S, and between 0.01mM and 0.04 mM for TlNO₃.

B. Light emitting bacteria, inducible by chlorinated chemicals

A5-23 and A5-24: chlorinated biphenyl biosensors:

Two colonies specifically emitting light on biphenyl were obtained.

These strains A5-23 and A5-24 still kept the feature to use biphenyl ascarbon source.

In the presence of biphenyl, some related aromatic compounds andtransformer-oil (askarel: marketed by the Company ACEC, Belgium) thesestrains elicit a strongly enhanced bioluminescence (FIG. 7).

The time-lag before bioluminescence increases can be shorteneddrastically in these strains by prior exposure of the bacteria to thecompounds of interest (pre-adaptation, i.e. pre-induction of certaingenes), probably because pre-adaptation (with the specific metal orxenobiotic to be further detected) provokes the synthesis of certainspecific gene products and is responsible for the beginning ofdegradation mechanisms or of resistance mechanisms.

This has been shown for the biphenyl biosensor (FIG. 8).

Biphenyl was used under the solid form. It has also been used in asolution of ethanol, such as the final concentration of biphenyl isbetween about 10 to about 500 ppm, dissolved in 0,5% (v/v) of ethanol.

Strain A5-23 can also be induced to produce light in the presence ofsome volatile chlorinated aliphatic solvents (di- and trichloroethane).The common denominator in these compounds and the aromatic inducers,mentioned above, is the presence of Cl atoms in all these molecules.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 883 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCAGTGCCAAGCTTGCATGCCTGCAGGTCGACATGGACGGTCCATGTGTCTTCCTTTCA60                ACGCAATATTGCGGACTCGGTCTGGTTTCTCGGCCTGTCGGTGGTCGTGGAGTCGTTTGA120               TCTGTTCCATGACGCCGGCGTCCTCCGGGCGGTCGTGACGCTTGCCTGATGCGGGCAACT180               CCGCATACACGTACAGCCATGCACTACGCTCACGTCTCCGATTCTGTTGTCGAGAGCATG240               CTCGTGATGCTGGTCGGCCTGATCGTCCTGTATGTGGGGTTCGCTGTCTATTTGCGGTGG300               AAGCATGGGCCCGCCCCCAAGCGTAAGACTGAGTAGGGGCGAAAGCGGCACCCCAAAACG360               AGCAGGCGAAAGCGATAATCGTAATCTGCTCTTAATGCTGGTGATCGAGGATTCATGTAA420               ACTTCGGCGAGCGCCCAGCCGTTAGTACTTCAACCCCAAGCCCCCCGCACAGTCTCCCAA480               GGAATGCGACGTTTCGTTCTGATCTTCGTGCTGCTCATTTGCCGTTCCAGTTTTCCTGGG540               CGGCAGCGGCACGCTATTGTCAGCACGAGAAAGCCACGGCCACTTGGCACCTTGGGCACC600               ACGAGCATCGTCATCAGCAGCCGGAAGGTAAAACGGATGCCGAGAAAAAGCCATTCGTGG660               ATACAGACTGCGGGGTATGCCATCTGGTCTCCCTCCCGTTCGTCTATGGACAGACGCAGG720               ACGTGTTGATAGCGAATCGGGTAGAAGTGACCGATACTCAACATTCGTCCGAGTTCTCGT780               CTCTGAATGCCAGGGCTCCCGACCGTCCTCAGTGGCAGCGTCTCGCTTGATCGGCGAGAC840               GACGACTCTTTTTCTCCTTTCGTCTCTCGCCGAATTCACTGGC883                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 883 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGGTCACGGTTCGAACGTACGGACGTCCAGCTGTACCTGCCAGGTACACAGAAGGAAAGT60                TGCGTTATAACGCCTGAGCCAGACCAAAGAGCCGGACAGCCACCAGCACCTCAGCAAACT120               AGACAAGGTACTGCGGCCGCAGGAGGCCCGCCAGCACTGCGAACGGACTACGCCCGTTGA180               GGCGTATGTGCATGTCGGTACGTGATGCGAGTGCAGAGGCTAAGACAACAGCTCTCGTAC240               GAGCACTACGACCAGCCGGACTAGCAGGACATACACCCCAAGCGACAGATAAACGCCACC300               TTCGTACCCGGGCGGGGGTTCGCATTCTGACTCATCCCCGCTTTCGCCGTGGGGTTTTGC360               TCGTCCGCTTTCGCTATTAGCATTAGACGAGAATTACGACCACTAGCTCCTAAGTACATT420               TGAAGCCGCTCGCGGGTCGGCAATCATGAAGTTGGGGTTCGGGGGGCGTGTCAGAGGGTT480               CCTTACGCTGCAAAGCAAGACTAGAAGCACGACGAGTAAACGGCAAGGTCAAAAGGACCC540               GCCGTCGCCGTGCGATAACAGTCGTGCTCTTTCGGTGCCGGTGAACCGTGGAACCCGTGG600               TGCTCGTAGCAGTAGTCGTCGGCCTTCCATTTTGCCTACGGCTCTTTTTCGGTAAGCACC660               TATGTCTGACGCCCCATACGGTAGACCAGAGGGAGGGCAAGCAGATACCTGTCTGCGTCC720               TGCACAACTATCGCTTAGCCCATCTTCACTGGCTATGAGTTGTAAGCAGGCTCAAGAGCA780               GAGACTTACGGTCCCGAGGGCTGGCAGGAGTCACCGTCGCAGAGCGAACTAGCCGCTCTG840               CTGCTGAGAAAAAGAGGAAAGCAGAGAGCGGCTTAAGTGACCG883                                __________________________________________________________________________

We claim:
 1. A fused gene comprising a promoter sequence derived from agene of Alcaligenes eutrophus strain CH34, SV661, DS185, AE453, or A5,said gene being a regulatory gene involved in the expression of eitherthe resistance to one or several metals or the catabolism of one orseveral xenobiotic compounds, said promoter from said regulatory genebeing an inducible promoter and is inducible in the presence of saidmetals or xenobiotic compounds, or both, and downstream of the promoter,a five gene lux (CDABE) operon said genes coding for subunit α and β ofluciferase, a fatty acid reductase, an acyltransferase and anacylprotein synthase, said operon being under the operational control ofsaid promoter, wherein at least one of said genes produces a detectablesignal.
 2. The fused gene according to claim 1, comprising the codingsequence of the gene(s) responsible for the resistance to one or severalmetal(s) or responsible for the catabolism of one or several xenobioticcompound(s).
 3. The fused gene according to claim 1, wherein the geneproducing a detectable signal:is either located downstream of thepromoter and upstream of the gene encoding the resistance or thecatabolism, or is located downstream of the promoter and downstream ofthe gene encoding the resistance or the catabolism, or is locateddownstream of the promoter and in the gene encoding the resistance orthe catabolism.
 4. The fused gene according to claim 1, wherein atermination sequence is located immediately upstream of the promoter. 5.The fused gene according to claim 1, wherein the gene producing adetectable signal is the lux operon originating from Vibrio fischeri,from Vibrio harveyi, from Photobacterium phosphoreum, or fromXenorhabdus luminescens.
 6. The fused gene according to claim 1, whereinthe gene encoding resistance to a metal or encoding the catabolism of axenobiotic compound originates from bacteria of the Alcaligeneseutrophus type.
 7. The fused gene according to claim 1, wherein thepromoter and the gene encoding resistance is a promoter and a geneencoding resistance to zinc that is obtained from plasmid pBR325comprising a czc fragment of plasmid pMOL30 from Alcaligenes eutrophusstrain CH34 and surrounding EcoRI fragment, digested with SalI, saidpromoter and gene encoding resistance is at the multiple cloning site ofthe plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M.,Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28, 1991,under n° I-1050), said plasmid comprising the lux operon of Vibriofischeri.
 8. The fused gene according to claim 1, wherein the promoterand the gene encoding resistance is the promoter and gene encodingresistance to cobalt that is obtained from plasmid pBR325 comprising aczc fragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34digested with EcoRI-PstI, said promoter and gene encoding resistance isat the multiple cloning site of the plasmid pUCD615 (in E. coli CM600deposited at the C.N.C.M., Institut Pasteur, 28 rue du Dr Roux, 75015Paris, on Feb. 28, 1991, under n° I-1050), said plasmid comprising thelux operon of Vibrio fischeri.
 9. A recombinant vector, for cloning orexpression, comprising a vector sequence, of the type plasmid, cosmid orphage and the fused gene according to claim 1, in one of thenonessential sites for its replication.
 10. The recombinant vectoraccording to claim 9, comprising in one of its nonessential sites forits replication, necessary elements to promote, in a cellular host,transcription and translation of the gene producing a detectable signaland transcription of the gene responsible for the resistance to a metalor responsible for the catabolism of a xenobiotic compound.
 11. Therecombinant vector according to claim 9, comprising necessary elementsto promote transcription and translation of the gene responsible for theresistance to a metal or responsible for catabolism of a xenobioticcompound.
 12. A cellular host comprising E. coli transformed by arecombinant vector according to claim 9 or comprising Alcaligeneseutrophus transconjugated by a recombinant vector according to claim 9,and comprising the regulation elements enabling the expression of thegene producing a detectable signal.
 13. The cellular host of claim 12,further comprising the regulation elements enabling the expression ofthe gene encoding resistance to a metal or of the gene encoding thecatabolism of a xenobiotic compound.
 14. A process for in vitropreparing a bacterial cellular host comprising a fused gene according toclaim 1, the process comprising the following steps:determining thepromoter derived from (a) gene(s) of Alcaligenes eutrophus and the geneencoding resistance to one or several heavy metals or encoding thecatabolism of one or several xenobiotic compound(s); isolating thecorresponding nucleic acid fragment of said promoter and gene; fusingsaid nucleic acid fragment with the lux (CDABE) operon deleted from itsown promoter; introducing the result of above-mentioned fusion into abacterial cellular host; and detecting light producing bacterialcellular host placed in an appropriate medium comprising one or severalmetal(s) or a xenobiotic compound.
 15. The process for in vitropreparing a bacterial cellular host of claim 14,wherein the promoter andthe gene encoding resistance to one or several heavy metals or encodingthe catabolism of one or several xenobiotic compounds(s) comprises amarker of the presence of the gene; wherein the lux operon furthercomprises a marker of the presence of the operon; and wherein theprocess further comprises selecting the cellular host with the marker(s)placed in a medium where the marker(s) can be detected.
 16. The processfor in vitro, preparing a bacterial cellular host of claim 15, whereinthe promoter is derived from an Alcaligenes eutrophus strain comprisingCH34, SV661, AE453, DS185, or A5.
 17. A process for preparing abacterial cellular host wherein said bacterial cellular host emits lightin the presence of zinc with a detection limit of 1 ppm and dynamicrange between 1 and 23 ppm Zinc comprising the steps of:(a) digestingwith SalI a plasmid pBR325 comprising a czc fragment of plasmid pMOL30from Alcaligenes eutrophus strain CH34 and surrounding EcoRI fragment toobtain a promoter and a gene encoding resistance to zinc; (b) insertingsaid promoter and said gene encoding resistance to zinc of step (a) intoa plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M., InstitutPasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, underNo. I-1050), at its multiple cloning site, and comprising the lux operonof Vibrio fischeri, to obtain a replicable plasmid; (c) transforming thereplicable plasmid of (b) in E. coli; (d) selecting the inserted plasmidon ampicillin plates with various concentrations of zinc; and (e)detecting the light producing E. coli in the presence of zinc.
 18. Aprocess for preparing a bacterial cellular host wherein said bacterialcellular host emits light in the presence of cobalt with a detectionlimit of 1 ppm and dynamic range between 1 and 23 ppm cobalt comprisingthe steps of:(a) digesting with SalI a plasmid pBR325 comprising a czcfragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34 andsurrounding EcoRI fragment to obtain a promoter and a gene encodingresistance to cobalt; (b) inserting said promoter and said gene encodingresistance to cobalt of step (a) into a plasmid pUCD615 (in E. coliCM600 deposited at the C.N.C.M. Institut Pasteur, 28 rue du DocteurRoux, 75015 Paris, on Feb. 28, 1991, under No. I-1050), at its multiplecloning site, and comprising the lux operon of Vibrio fischeri, toobtain a replicable plasmid; (c) transforming the replicable plasmid of(b) in E. coli; (d) selecting the inserted plasmid on ampicillin plateswith various concentrations of cobalt; and (e) detecting the lightproducing E. coli in the presence of cobalt.
 19. A process for in vivopreparing a bacterial cellular host comprising a fused gene according toclaim 1, comprising the following steps:conjugating, to obtaintransconjugants, a cellular host comprising a promoter derived fromAlcaligenes eutrophus and a gene encoding the resistance to a metal orencoding the catabolism of a xenobiotic compound with another cellularhost comprising a transposon comprising the lux (CDABE) operon withoutits own promoter; recovering the transconjugants; and selectingtransconjugants emitting light in the presence of a medium containing ametal or a xenobiotic compound.
 20. The process for in vivo preparing abacterial cellular host comprising a fused gene according to claim 19further comprising the step of:applying transconjugants to media withdifferent concentrations of metal or xenobiotics.
 21. The process for invivo preparing a bacterial cellular host comprising a fused geneaccording to claim 19, wherein:the promoter and gene encoding theresistance to metal or encoding the catabolism of xenobiotic compoundfurther comprises a marker for the presence of the gene; the transposoncomprising lux operon further comprises a marker for the presence ofsaid gene; and the process further comprises selecting transconjugantswith the marker(s) placed in a medium where the marker(s) can bedetected.
 22. The process according to claim 19, wherein:the bacterialcellular host comprising a promoter and a gene encoding the resistanceto a metal is Alcaligenes eutrophus and the cellular host comprising atransposon is E. coli comprising the vector pUCD623, itself comprising atransposon Tn4431 which is Ty21 transposon comprising the tetracyclineresistance and the lux operon of Vibrio fischeri without its ownpromoter, and the process further comprises the steps of:selecting thetransconjugants on tetracycline plates; replicating the transconjugantson media with different concentrations of metals; and detecting lightproducing transconjugants.
 23. The process according to claim 22 forpreparing a bacterial cellular host emitting light in the presence ofchromium with a detection limit of 1 ppm of chromium, wherein thecellular host comprising a promoter and a gene resistant to metal isAlcaligenes eutrophus SV661 (deposited at the C.N.C.M., InstitutPasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28, 1991, under n°I-1046) and the cellular host comprising the transposon is E. colistrain CM601 (deposited at the C.N.C.M., Institut Pasteur, 28 rue du DrRoux, 75015 Paris, on Feb. 28, 1991, under n° I-1051), which givesstrain AE714, transferred into strain A5.3, which is a rifampicin mutantof the biphenyl degrading A5 strain (I-1047), to give strain AE859,which gives light expression in the presence of chromium.
 24. Theprocess according to claim 22, for preparing a bacterial cellular hostemitting light in the presence of a concentration of at least 1 ppm ofnickel, wherein the cellular host comprising a promoter and a generesistant to a metal is Alcaligenes eutrophus AE453 and the cellularhost comprising the transposon is E. coli strain CM601 (deposited at theC.N.C.M., Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28,1991, under n° I-1051), which gives strain AE453 (deposited at theC.N.C.M., Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28,1991, under n° I-1049), transferred into strain A5.3, which is arifampicin mutant of the biphenyl degrading A5 strain (I-1047), to givestrain AE891, which gives light expression in the presence of nickel.25. The process according to claim 22, for preparing a bacterialcellular host emitting light in the presence of copper, wherein thecellular host comprising a promoter and a gene resistant to a metal isAlcaligenes eutrophus DS185 (deposited at the C.N.C.M., InstitutPasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28, 1991, under n°I-1048), and the cellular host comprising the transposon is E. colistrain CM601 (deposited at the C.N.C.M., Institut Pasteur, 28 rue du DrRoux, 75015 Paris, on Feb. 28, 1991, under n° I-1051), which givesAE866, which gives light expression in the presence of copper.
 26. Theprocess according to claim 22, for preparing a bacterial cellular hostemitting light in the presence of copper, wherein the cellular hostcomprising a promoter and a gene resistant to a metal is Alcaligeneseutrophus DS310, and the cellular host comprising the transposon is E.coli strain CM601 (deposited at the C.N.C.M., Institut Pasteur, 28 ruedu Dr Roux, 75015 Paris, on Feb. 28, 1991, under n° I-1051), which givesAE890, which gives light expression in the presence of copper and cannotgrow on minimal plates containing lead.
 27. The process according toclaim 22, for preparing a cellular host emitting light in the presenceof biphenyl, wherein the cellular host comprising a promoter and a geneencoding the catabolism of biphenyl compounds is Alcaligenes eutrophusA5 (deposited at the C.N.C.M., Institut Pasteur, 28 rue du Dr Roux,75015 Paris, on Feb. 28, 1991, under n° I-1047), and the cellular hostcomprising the transposon is E. coli strain CM601 (deposited at theC.N.C.M., Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, on Feb. 28,1991, under n° I-1051), which gives A5.23 or A5.24, which gives lightexpression in the presence of biphenyl compounds.
 28. A cellular hostprepared according to the process of claim
 15. 29. A cellular hostprepared according to the process of claim
 19. 30. A process fordetecting, in a liquid medium, a metal or a xenobiotic compound in aconcentration range of about 1 to about 120 ppm, comprising the stepsof:placing a cellular host of claim 9 which has been lyophilized andimmobilized on a solid support into a liquid medium to form a liquidculture medium; introducing a sample of said liquid culture mediumcontaining a cellular host of claim 9 into a sample taken from a liquidmedium, in which the presence of a metal or of a xenobiotic compound isto be detected; and detecting the signal generated by the presence ofsaid metal or the presence of said xenobiotic compound by detectingmeans.
 31. The process for detecting a metal or xenobiotic compoundaccording to claim 30, wherein the signal is light and the detectingmeans is a luminometer.
 32. A process for detecting, in a liquid medium,a metal or a xenobiotic compound in a concentration range of about 1 toabout 120 ppm, comprising the steps of:introducing a cellular host ofclaim 12 contained in a liquid culture medium, into a sample taken froma liquid medium; and detecting the signal generated by the presence ofsaid metal or in the presence of xenobiotic compound by detecting means.33. The process for detecting a metal or xenobiotic compound accordingto claim 32, wherein the liquid medium is an aqueous medium, the signalis light, and the detecting means is a luminometer.
 34. A kit fordetecting a metal or xenobiotic compound in a concentration at leastabout 1 ppm for metals and as little as 1 ppb for xenobiotics,comprising:a cellular host of claim 12; and detection means to detectthe signal generated by the presence of said metal or xenobioticcompound.
 35. The kit according to claim 34, wherein the detection meansdetects light.